Abstract
The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.
Highlights
Sepsis commonly causes severe organ failures including acute lung injury (ALI) and results in high mortality and substantial morbidity
We previously described that compared with macromolecular LPS, the Lipid A fraction of LPS induces a discrete MAP kinases (MAPKs) activation (ERK activation) in murine acute lung injury [8]
Leukotriene biosynthesis inhibitor enhances ERK activation after Lipid A and LPS stimulation To determine the mechanism of leukotriene biosynthesis inhibition on LPS and Lipid A mediated cytokine expression protein expression ERK (p44/42), 5-lipoxygenase (5-LO) and cyclooxygenase 2(COX-2) were quantified in cell lysates by immunoblotting
Summary
Sepsis commonly causes severe organ failures including acute lung injury (ALI) and results in high mortality and substantial morbidity. Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria (GNB) [3] is recognized as a key to the pathogenesis of GNB-associated sepsis. Recognition of bacterial pathogen-associated molecular patterns (PAMPs) such as LPS, by macrophage toll-like receptors is a key component of host defences against infection by Gram-negative bacteria [4]. Macromolecular LPS binds monocyte cell-surface expressed PAMPS including TLR-4 and -2, resulting in MyD88-dependent signaling and downstream inflammatory cascade activation via phospohoactivation of IkB kinase (IKK)-b and MAP kinases (MAPKs). This in turn mediates NF-kB and AP-1 dependent nuclear transcription of pro-inflammatory cytokines including IL-1 and TNFa [7]
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